Microneedle array delivery of Yersinia pestis recapitulates bubonic plague.

Fleas transmit Yersinia pestis directly within the dermis of mammals to cause bubonic plague. Syringe-mediated inoculation is widely used to recapitulate bubonic plague and study Y. pestis pathogenesis. However, intradermal needle inoculation is tedious, error prone, and poses a significant safety risk for laboratorians. Microneedle arrays (MNAs) are micron-scale polymeric structures that deliver materials to the dermis, while minimizing the risk of needle sticks. We demonstrated that MNA inoculation is a viable strategy to recapitulate bubonic plague and study bacterial virulence by defining the parameters needed to establish a lethal infection in the mouse model and characterizing the course of infection using live-animal optical imaging. Using MNAs, we also demonstrated that Y. pestis must overcome calprotectin-mediated zinc restriction within the dermis and dermal delivery of an attenuated mutant has vaccine potential. Together, these data demonstrate that MNAs are a safe alternative to study Y. pestis pathogenesis in the laboratory.

Type 3 secretion system induced leukotriene B4 synthesis by leukocytes is actively inhibited by Yersinia pestis to evade early immune recognition.

Subverting the host immune response to inhibit inflammation is a key virulence strategy of Yersinia pestis. The inflammatory cascade is tightly controlled via the sequential action of lipid and protein mediators of inflammation. Because delayed inflammation is essential for Y. pestis to cause lethal infection, defining the Y. pestis mechanisms to manipulate the inflammatory cascade is necessary to understand this pathogen's virulence. While previous studies have established that Y. pestis actively inhibits the expression of host proteins that mediate inflammation, there is currently a gap in our understanding of the inflammatory lipid mediator response during plague. Here we used the murine model to define the kinetics of the synthesis of leukotriene B4 (LTB4), a pro-inflammatory lipid chemoattractant and immune cell activator, within the lungs during pneumonic plague. Furthermore, we demonstrated that exogenous administration of LTB4 prior to infection limited bacterial proliferation, suggesting that the absence of LTB4 synthesis during plague contributes to Y. pestis immune evasion. Using primary leukocytes from mice and humans further revealed that Y. pestis actively inhibits the synthesis of LTB4. Finally, using Y. pestis mutants in the Ysc type 3 secretion system (T3SS) and Yersinia outer protein (Yop) effectors, we demonstrate that leukocytes recognize the T3SS to initiate the rapid synthesis of LTB4. However, several Yop effectors secreted through the T3SS effectively inhibit this host response. Together, these data demonstrate that Y. pestis actively inhibits the synthesis of the inflammatory lipid LTB4 contributing to the delay in the inflammatory cascade required for rapid recruitment of leukocytes to sites of infection.

Droplet Tn-Seq identifies the primary secretion mechanism for yersiniabactin in Yersinia pestis.

Nutritional immunity includes sequestration of transition metals from invading pathogens. Yersinia pestis overcomes nutritional immunity by secreting yersiniabactin to acquire iron and zinc during infection. While the mechanisms for yersiniabactin synthesis and import are well-defined, those responsible for yersiniabactin secretion are unknown. Identification of this mechanism has been difficult because conventional mutagenesis approaches are unable to inhibit trans-complementation by secreted factors between mutants. To overcome this obstacle, we utilized a technique called droplet Tn-seq (dTn-seq), which uses microfluidics to isolate individual transposon mutants in oil droplets, eliminating trans-complementation between bacteria. Using this approach, we first demonstrated the applicability of dTn-seq to identify genes with secreted functions. We then applied dTn-seq to identify an AcrAB efflux system as required for growth in metal-limited conditions. Finally, we showed this efflux system is the primary yersiniabactin secretion mechanism and required for virulence during bubonic and pneumonic plague. Together, these studies have revealed the yersiniabactin secretion mechanism that has eluded researchers for over 30 years and identified a potential therapeutic target for bacteria that use yersiniabactin for metal acquisition.

Crystal Structure and Twisted Aggregates of Oxcarbazepine Form III.

Polymorphism and crystal habit play vital roles in dictating the properties of crystalline materials. Here, the structure and properties of oxcarbazepine (OXCBZ) form III are reported along with the occurrence of twisted crystalline aggregates of this metastable polymorph. OXCBZ III can be produced by crystallization from the vapor phase and by recrystallization from solution. The crystallization process used to obtain OXCBZ III is found to affect the pitch, with the most prominent effect observed from the sublimation-grown OXCBZ III material where the pitch increases as the length of aggregates increases. Sublimation-grown OXCBZ III follows an unconventional mechanism of formation with condensed droplet formation and coalescence preceding nucleation and growth of aggregates. A crystal structure determination of OXCBZ III from powder X-ray diffraction methods, assisted by crystal structure prediction (CSP), reveals that OXCBZ III, similar to carbamazepine form II, contains void channels in its structure with the channels, aligned along the c crystallographic axis, oriented parallel to the twist axis of the aggregates. The likely role of structural misalignment at the lattice or nanoscale is explored by considering the role of molecular and closely related structural impurities informed by crystal structure prediction.

Packing Preferences of Chalcones: A Model Conjugated Pharmaceutical Scaffold.

We sought the crystal packing preferences of the chalcone scaffold by analyzing 232 single-component crystal structures of chalcones with a small (six or fewer non-hydrogen atoms) substituent on either or both rings, including the unsubstituted molecule. This covers 216 molecules, as some are polymorphic, and 277 independent molecular conformations, as 16% of the crystal structures have more than one symmetry independent molecule. Quantum mechanical conformational profiles of the unsubstituted molecule and the almost 5000 crystal structures within 20 kJ mol-1 of the global minimum generated in a crystal structure prediction (CSP) study have been used to complement this analysis. Although π conjugation would be expected to favor a planar molecule, there are a significant number of crystal structures containing nonplanar molecules with an approximately 50° angle between the aromatic rings. The relative orientations of the molecules in the inversion-related dimers and translation-related dimers in the experimental crystal structures show the same trends as in the CSP-generated structures for the unsubstituted molecule, allowing for the substituent making the side-to-side distances larger. There is no type of dimer geometry associated with particularly favorable lattice energies for the chalcone core. Less than a third of the experimental structures show a face-to-face contact associated with π···π stacking. Analysis of the experimental crystal structures with XPac and Mercury finds various pairs of isostructural crystals, but the largest isostructural set had only 15 structures, with all substituents (mainly halogens) in the para position. The most common one-dimensional motif, found in half of the experimental crystal structures, is a translation-related side-to-side packing, which can be adopted by all the observed conformations. This close-packed motif can be adopted by chalcones with a particularly wide variety of substituents as the substituents are at the periphery. Thus, although the crystal structures of the substituted chalcones show thermodynamically plausible packings of the chalcone scaffold, there is little evidence for any crystal engineering principle of preferred chalcone scaffold packing beyond close packing of the specific molecule.

Progress in understanding crystallisation: a personal perspective.

After this Discussion meeting, most participants felt that we do not understand crystallisation. However, in the 1980s, I believe that most scientists would have considered that crystallisation was adequately understood. These concluding remarks give a personal impression of the progress that has been made towards appreciating the complexity of crystallisation over the past forty years.

Fluorescent sensors of siderophores produced by bacterial pathogens.

Siderophores are iron-chelating molecules that solubilize Fe3+ for microbial utilization and facilitate colonization or infection of eukaryotes by liberating host iron for bacterial uptake. By fluorescently labeling membrane receptors and binding proteins, we created 20 sensors that detect, discriminate, and quantify apo- and ferric siderophores. The sensor proteins originated from TonB-dependent ligand-gated porins (LGPs) of Escherichia coli (Fiu, FepA, Cir, FhuA, IutA, BtuB), Klebsiella pneumoniae (IroN, FepA, FyuA), Acinetobacter baumannii (PiuA, FepA, PirA, BauA), Pseudomonas aeruginosa (FepA, FpvA), and Caulobacter crescentus (HutA) from a periplasmic E. coli binding protein (FepB) and from a human serum binding protein (siderocalin). They detected ferric catecholates (enterobactin, degraded enterobactin, glucosylated enterobactin, dihydroxybenzoate, dihydroxybenzoyl serine, cefidericol, MB-1), ferric hydroxamates (ferrichromes, aerobactin), mixed iron complexes (yersiniabactin, acinetobactin, pyoverdine), and porphyrins (hemin, vitamin B12). The sensors defined the specificities and corresponding affinities of the LGPs and binding proteins and monitored ferric siderophore and porphyrin transport by microbial pathogens. We also quantified, for the first time, broad recognition of diverse ferric complexes by some LGPs, as well as monospecificity for a single metal chelate by others. In addition to their primary ferric siderophore ligands, most LGPs bound the corresponding aposiderophore with ∼100-fold lower affinity. These sensors provide insights into ferric siderophore biosynthesis and uptake pathways in free-living, commensal, and pathogenic Gram-negative bacteria.

Siderophore-mediated zinc acquisition enhances enterobacterial colonization of the inflamed gut.

Zinc is an essential cofactor for bacterial metabolism, and many Enterobacteriaceae express the zinc transporters ZnuABC and ZupT to acquire this metal in the host. However, the probiotic bacterium Escherichia coli Nissle 1917 (or "Nissle") exhibits appreciable growth in zinc-limited media even when these transporters are deleted. Here, we show that Nissle utilizes the siderophore yersiniabactin as a zincophore, enabling Nissle to grow in zinc-limited media, to tolerate calprotectin-mediated zinc sequestration, and to thrive in the inflamed gut. We also show that yersiniabactin's affinity for iron or zinc changes in a pH-dependent manner, with increased relative zinc binding as the pH increases. Thus, our results indicate that siderophore metal affinity can be influenced by the local environment and reveal a mechanism of zinc acquisition available to commensal and pathogenic Enterobacteriaceae.

Yersiniabactin contributes to overcoming zinc restriction during Yersinia pestis infection of mammalian and insect hosts.

Yersinia pestis causes human plague and colonizes both a mammalian host and a flea vector during its transmission cycle. A key barrier to bacterial infection is the host's ability to actively sequester key biometals (e.g., iron, zinc, and manganese) required for bacterial growth. This is referred to as nutritional immunity. Mechanisms to overcome nutritional immunity are essential virulence factors for bacterial pathogens. Y. pestis produces an iron-scavenging siderophore called yersiniabactin (Ybt) that is required to overcome iron-mediated nutritional immunity and cause lethal infection. Recently, Ybt has been shown to bind to zinc, and in the absence of the zinc transporter ZnuABC, Ybt improves Y. pestis growth in zinc-limited medium. These data suggest that, in addition to iron acquisition, Ybt may also contribute to overcoming zinc-mediated nutritional immunity. To test this hypothesis, we used a mouse model defective in iron-mediated nutritional immunity to demonstrate that Ybt contributes to virulence in an iron-independent manner. Furthermore, using a combination of bacterial mutants and mice defective in zinc-mediated nutritional immunity, we identified calprotectin as the primary barrier for Y. pestis to acquire zinc during infection and that Y. pestis uses Ybt to compete with calprotectin for zinc. Finally, we discovered that Y. pestis encounters zinc limitation within the flea midgut, and Ybt contributes to overcoming this limitation. Together, these results demonstrate that Ybt is a bona fide zinc acquisition mechanism used by Y. pestis to surmount zinc limitation during the infection of both the mammalian and insect hosts.

Toward Physics-Based Solubility Computation for Pharmaceuticals to Rival Informatics.

We demonstrate that physics-based calculations of intrinsic aqueous solubility can rival cheminformatics-based machine learning predictions. A proof-of-concept was developed for a physics-based approach via a sublimation thermodynamic cycle, building upon previous work that relied upon several thermodynamic approximations, notably the 2RT approximation, and limited conformational sampling. Here, we apply improvements to our sublimation free-energy model with the use of crystal phonon mode calculations to capture the contributions of the vibrational modes of the crystal. Including these improvements with lattice energies computed using the model-potential-based Ψmol method leads to accurate estimates of sublimation free energy. Combining these with hydration free energies obtained from either molecular dynamics free-energy perturbation simulations or density functional theory calculations, solubilities comparable to both experiment and informatics predictions are obtained. The application to coronene, succinic acid, and the pharmaceutical desloratadine shows how the methods must be adapted for the adoption of different conformations in different phases. The approach has the flexibility to extend to applications that cannot be covered by informatics methods.

A non-empirical intermolecular force-field for trinitrobenzene and its application in crystal structure prediction.

An anisotropic atom-atom distributed intermolecular force-field (DIFF) for rigid trinitrobenzene (TNB) is developed using distributed multipole moments, dipolar polarizabilities, and dispersion coefficients derived from the charge density of the isolated molecule. The short-range parameters of the force-field are fitted to first- and second-order symmetry-adapted perturbation theory dimer interaction energy calculations using the distributed density-overlap model to guide the parameterization of the short-range anisotropy. The second-order calculations are used for fitting the damping coefficients of the long-range dispersion and polarization and also for relaxing the isotropic short-range coefficients in the final model, DIFF-srL2(rel). We assess the accuracy of the unrelaxed model, DIFF-srL2(norel), and its equivalent without short-range anisotropy, DIFF-srL0(norel), as these models are easier to derive. The model potentials are contrasted with empirical models for the repulsion-dispersion fitted to organic crystal structures with multipoles of iterated stockholder atoms (ISAs), FIT(ISA,L4), and with Gaussian Distributed Analysis (GDMA) multipoles, FIT(GDMA,L4), commonly used in modeling organic crystals. The potentials are tested for their ability to model the solid state of TNB. The non-empirical models provide more reasonable relative lattice energies of the three polymorphs of TNB and propose more sensible hypothetical structures than the empirical force-field (FIT). The DIFF-srL2(rel) model successfully has the most stable structure as one of the many structures that match the coordination sphere of form III. The neglect of the conformational flexibility of the nitro-groups is a significant approximation. This methodology provides a step toward force-fields capable of representing all phases of a molecule in molecular dynamics simulations.

Diabat method for polymorph free energies: Extension to molecular crystals.

Lattice-switch Monte Carlo and the related diabat methods have emerged as efficient and accurate ways to compute free energy differences between polymorphs. In this work, we introduce a one-to-one mapping from the reference positions and displacements in one molecular crystal to the positions and displacements in another. Two features of the mapping facilitate lattice-switch Monte Carlo and related diabat methods for computing polymorph free energy differences. First, the mapping is unitary so that its Jacobian does not complicate the free energy calculations. Second, the mapping is easily implemented for molecular crystals of arbitrary complexity. We demonstrate the mapping by computing free energy differences between polymorphs of benzene and carbamazepine. Free energy calculations for thermodynamic cycles, each involving three independently computed polymorph free energy differences, all return to the starting free energy with a high degree of precision. The calculations thus provide a force field independent validation of the method and allow us to estimate the precision of the individual free energy differences.

Calculation of Diamagnetic Susceptibility Tensors of Organic Crystals: From Coronene to Pharmaceutical Polymorphs.

Understanding why crystallization in strong magnetic fields can lead to new polymorphs requires methods to calculate the diamagnetic response of organic molecular crystals. We develop the calculation of the macroscopic diamagnetic susceptibility tensor, χcryst, for organic molecular crystals using periodic density functional methods. The crystal magnetic susceptibility tensor, χcryst, for all experimentally known polymorphs, and its molecular counterpart, χmol, are calculated for flexible pharmaceuticals such as carbamazepine, flufenamic acid, and chalcones, and rigid molecules, such as benzene, pyridine, acridine, anthracene, and coronene, whose molecular magnetic properties have been traditionally studied. A tensor addition method is developed to approximate the crystal diamagnetic susceptibility tensor, χcryst, from the molecular one, χmol, giving good agreement with those calculated directly using the more costly periodic density functional method for χcryst. The response of pharmaceutical molecules and crystals to magnetic fields, as embodied by χcryst, is largely determined by the packing in the crystal, as well as the molecular conformation. The anisotropy of χcryst can vary considerably between polymorphs though the isotropic terms are fairly constant. The implications for developing a computational method for predicting whether crystallization in a magnetic field could produce a novel or different polymorph are discussed.

A Prolific Solvate Former, Galunisertib, under the Pressure of Crystal Structure Prediction, Produces Ten Diverse Polymorphs.

The solid form screening of galunisertib produced many solvates, prompting an extensive investigation into possible risks to the development of the favored monohydrate form. Inspired by crystal structure prediction, the search for neat polymorphs was expanded to an unusual range of experiments, including melt crystallization under pressure, to work around solvate formation and the thermal instability of the molecule. Ten polymorphs of galunisertib were found; however, the structure predicted to be the most stable has yet to be obtained. We present the crystal structures of all ten unsolvated polymorphs of galunisertib, showing how state-of-the-art characterization methods can be combined with emerging computational modeling techniques to produce a complete structure landscape and assess the risk of late-appearing, more stable polymorphs. The exceptional conformational polymorphism of this prolific solvate former invites further development of methods, computational and experimental, that are applicable to larger, flexible molecules with complex solid form landscapes.

Charge Distributions of Nitro Groups Within Organic Explosive Crystals: Effects on Sensitivity and Modeling.

The charge distribution of NO2 groups within the crystalline polymorphs of energetic materials strongly affects their explosive properties. We use the recently introduced basis-space iterated stockholder atom partitioning of high-quality charge distributions to examine the approximations that can be made in modeling polymorphs and their physical properties, using 1,3,5-trinitroperhydro-1,3,5-triazine, trinitrotoluene, 1-3-5-trinitrobenzene, and hexanitrobenzene as exemplars. The NO2 charge distribution is strongly affected by the neighboring atoms, the rest of the molecules, and also significantly by the NO2 torsion angle within the possible variations found in observed crystal structures. Thus, the proposed correlations between the molecular electrostatic properties, such as trigger-bond potential or maxima in the electrostatic potential, and impact sensitivity will be affected by the changes in conformation that occur on crystallization. We establish the relationship between the NO2 torsion angle and the likelihood of occurrence in observed crystal structures, the conformational energy, and the charge and dipole magnitude on each atom, and how this varies with the neighboring groups. We examine the effect of analytically rotating the atomic multipole moments to model changes in torsion angle and establish that this is a viable approach for crystal structures but is not accurate enough to model the relative lattice energies. This establishes the basis of transferability of the NO2 charge distribution for realistic nonempirical model intermolecular potentials for simulating energetic materials.

Control and prediction of the organic solid state: a challenge to theory and experiment†.

The ability of theoretical chemists to quantitatively model the weak forces between organic molecules is being exploited to predict their crystal structures and estimate their physical properties. Evolving crystal structure prediction methods are increasingly being used to aid the design of organic functional materials and provide information about thermodynamically plausible polymorphs of speciality organic materials to aid, for example, pharmaceutical development. However, the increasingly sophisticated experimental studies for detecting the range of organic solid-state behaviours provide many challenges for improving quantitative theories that form the basis for the computer modelling. It is challenging to calculate the relative thermodynamic stability of different organic crystal structures, let alone understand the kinetic effects that determine which polymorphs can be observed and are practically important. However, collaborations between experiment and theory are reaching the stage of devising experiments to target the first crystallization of new polymorphs or create novel organic molecular materials.